A decellularized human corneal scaffold for anterior corneal surface reconstruction

Abstract

Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction

“…However, significant drawbacks were the reported long elution times required for the removal of cellular components (~4–7 days). In the current study, we validated our recently published rapid decellularization protocol [ 24 ] for efficient decellularization of limbal tissue. Our data show that 1% SD with DNAse and 4% dextran allows for fast (~1 day) and efficient decellularization of limbal tissue with complete removal of cellular components (HLA-ABC and nuclear debris).…”

“…Various protocols have been used to decellularize human corneal tissue [ 22 , 23 , 24 ], but are limited to very few studies on human limbal tissue so far [ 20 , 21 ]. In these studies, combinations of SD or hypertonic salt (NaCl) and nucleases have been used to decellularize the human corneal limbus.…”

“…Our data show that 1% SD with DNAse and 4% dextran allows for fast (~1 day) and efficient decellularization of limbal tissue with complete removal of cellular components (HLA-ABC and nuclear debris). Thus, the rapid decellularization protocol can be extended to treat limbal tissue with similar efficiency as corneal scaffolds [ 24 ]. The lack of nuclear and cellular material in decellularized tissues reduces the antigenicity of the limbal scaffolds and could therefore provide nonimmunogenic scaffolds for LEPC transplantation to treat LSCD.…”

“…Most protocols suggested for decellularization are very time consuming (~4–7 days) [ 20 , 21 ]. In a previous study, we reported on a fast and efficient method (~1 day) to decellularize the human cornea using sodium deoxycholate (SD), allowing for the preservation of mechanical, optical, and biochemical properties as well as the cellular biocompatibility of the central cornea (~6 to 7 mm diameter central cornea) [ 24 ]. These corneal scaffolds may serve to reconstruct lamellar structural defects of the central cornea.…”

“…In contrast, the current manuscript aims to provide a scaffold for limbal stem cell transplantation with the long-term goal to treat patients suffering from structural damage in the limbal stem cell niche. The limbal stem cell niche is very different from the central cornea [ 2 , 3 , 25 ] studied in the previous report [ 24 ] as it is endowed with crypt structures, fine stromal protrusions, a particular ECM composition, and a basement membrane (BM) composition, which regulates LEPC homeostasis [ 26 , 27 ]. In light of these major structural and biological distinctions, a focused assessment of a decellularization protocol in limbal tissue is warranted.…”